Biopolymer produced by fermenting the extract of soybean with folic acid and a composition containing thereof

ABSTRACT

Provided are a folyl extract of fermented soybean (EFS) produced by fermenting a culture including a folic acid and soybean extract by using a microorganism, and a composition including the folyl EFS. The folyl EFS has an anti-histamine effect, an anti-allergic effect, a calcium-absorption-promotion effect, a bone-growth-promotion effect, a cell growth promotion effect, a collagen biosynthesis promotion effect, a wrinkle improvement effect, and an UV-induced cell damage inhibition effect. Accordingly, the folyl EFS can be used in a skin external application or cosmetic composition, a health supplement food composition, a feed composition, and a pharmaceutical composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationNo. PCT/KR2008/005816, filed Oct. 2, 2008 and published in English onApr. 9, 2009 as WO 2009/045064, which claims the benefit of KoreanPatent Application No. 10-2007-0100311, filed Oct. 5, 2007, both ofwhich are hereby incorporated by reference to the extent notinconsistent with the disclosure herewith.

TECHNICAL FIELD

The present invention relates to a folyl extract of fermented soybean(EFS) produced by fermenting a culture including a folic acid andsoybean extract by using a microorganism, and a composition includingthe same.

The folyl EFS or composition according to the present invention has ananti-histamine effect, an anti-allergic effect, a calcium absorptionacceleration effect, a bone growth acceleration effect, a cell growthacceleration effect, a collagen biosynthesis acceleration effect, awrinkle-improvement effect, and an effect of inhibiting cell damagecaused by ultra-violet (UV) radiation. Accordingly, the folyl EFS orcomposition according to the present invention can be used in skinexternal applications or cosmetic compositions, health supplement foodcompositions, feed compositions, or pharmaceutical compositions.

BACKGROUND ART

Chungkookjang and Natto are representative fermented foods and areproduced by fermenting soybeans using Bacillus subtilis. When soybeansare fermented, γ-polyglutamate and polyfructan polymers are generated.γ-polyglutamate is known to have moisturizing and cell activationfunctions, and when used together with an anti-cancer drug such asTaxol, the effect of the drug used is increased. Polyfructan is adietary fiber that is used to lose weight and when administered, gastricfullness and appetite suppression can occur and thus, obesity can beprevented. In addition, it is reported that polyfructan inhibits growthof harmful cells in bowels and helps Bifidobacterium grow (see Journalof Applied Microbiology, 92, 5, p 958-963).

Patents related to fermented soybeans include a patent related to acosmetic material (see Korean Registration Patent No. 0451631), and apatent related to a skin external application (see Korean PublicationPatent No. 2001-0083876). Seong mun-hoe et al. developed an enzyme-basedtechnique for producing γ-polyglutamate having a molecular weight of2,000 kDa or more (see App. & Environ. Microbiol. 2004; 70(7),4249-4255; and Korean Registration Patent No. 10-0399091). Also, theyregistered a patent based on research results regarding a material inwhich an ascorbic acid binds to γ-polyglutamate having such a highmolecular weight (Korean Registration Patent No. 10-0485727). However, anovel type of fermented soybean polymer in which a folic acid (alsocalled as vitamin B₉) binds to γ-polyglutamate and an applicationthereof have not been developed.

Folic acid is known to be a material that lowers levels of homocysteinethat is associated with heart disease and dementia of elderly people.Accordingly, when folic acid is used in foods for elderly people, higheffects can be obtained. Folic acid naturally exists in a polyglutamateform and thus can be easily absorbed into bodies of animals and humans.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of microscopic images showing an anti-histamineeffect of a folyl extract of fermented soybean (folyl EFS), wherein FIG.1A shows a microscopic image of normal cells, FIG. 1B shows amicroscopic image of cells 30 minutes after the cells were treated witha stimulant, and FIG. 1C shows a microscopic image of cells 30 minutesafter the cells were treated with a folyl EFS and a stimulus;

FIG. 2 is a bar chart of test results showing ahistamine-secretion-inhibition effect of a folyl EFS by quantifyinghistamine and cromolyn that is known as a therapeutic agent for asthma;

FIG. 3 is a bar chart showing test results of the folyl EFS with respectto anaphylaxis, represented in a death rate of each group when astimulus is intraperperitoneally injected;

FIG. 4 is a bar chart of test results showing acalcium-absorption-promotion effect of a folyl EFS in small bowels ofanimals, wherein, in an experiment to obtain the results, the animalsare given the folyl EFS added feed, small bowels of the animals wereseparated and calcium of each group was quantified, and the quantifiedresults were represented in a ratio with respect to a group givennothing added feed;

FIG. 5 is a bar chart of test results showing a growth promotion effectof a folyl EFS, wherein, in an experiment to obtain the results, ratswere fed the folyl EFS for a predetermined period of time, blood wastaken from each group, and IGF-1 in the blood was quantified by ELISA;

FIG. 6 shows microscopic images of the primary culture of a fibroblastwith a folyl EFS or a folic acid, after being incubated in a fibroblastculture medium (FGM, KDR Co.) for 24 hours;

FIG. 7 is a bar chart showing quantification results of viable cellsthrough neutral red (NR) assay after a primary culture of a fibroblastwith a folyl EFSor a folic acid, after being incubated in a fibroblastgrowth medium (FGM, KDR Co.) for 24 hours; and

FIG. 8 is a bar chart of results showing a collagen biosynthesispromotion effect of a folyl EFS, wherein, in an experiment to obtain theresults, collagen biosynthesis was promoted using a primary culture of afibroblast and the collagen was quantified by ELISA, and the collagenbiosynthesis promotion effect of the folyl EFS was compared with thecollagen biosynthesis promotion effect of TGF-β that is known as acollagen biosynthesis promotion material; and

FIG. 9 is a bar chart showing quantification results of viable cellsthrough NR assay, wherein, in an experiment to obtain the results, aprimary culture of human keratinocyte was incubated with a folyl EFS inwhich the content of folic acid was about 10 wt. % in a humankeratinocyte growth medium (KGM, KDR Co.) for about 72 hours,ultra-violet (UV) rays having a wavelength of 306 nm were irradiated togrown cells, and then incubated for about 24 hours.

DETAILED DESCRIPTION OF THE INVENTION Technical Problem

The present invention provides a novel type of fermented soybean thathas excellent characteristics, is inexpensive, and can be used invarious applications.

Technical Solution

To achieve this, the inventors of the present invention tried to fermenta composition including a folic acid and soybean extract and produced anovel folyl extract of fermented soybean (folyl EFS).

Advantageous Effects

A folyl extract of fermented soybean (folyl EFS) according to thepresent invention inhibits mast cells from generating histamine, whereinthe phenomenon in which mast cells generate histamine is one of thesymptoms occurring in people who have serious cutaneous adversereactions such as allergy. Also, the folyl EFS is very effective foralleviating anaphylaxis. In addition, the folyl EFS promotes absorptionof minerals such as calcium in the small bowel, and promotes femurs(thigh bones) growth of mammals. Furthermore, the folyl EFS promotesbiosynthesis of collagen due to its cell activation function and thus,when applied to human skin, a wrinkle-improvement effect can be obtainedand thus, various other effects, such as moisturizing and anti-allergiceffects, can also be obtained. Also, the folyl EFS protects cells frombeing damaged by ultra-violet (UV) radiation. Accordingly, the folyl EFSand a composition including the folyl EFS can be used in healthsupplement foods, cosmetics, skin external applications, pharmaceuticalproducts, and livestock feeds. Also, the folyl EFS according to thepresent invention can be produced at lower costs and in higher yieldsthan other products having similar effects.

MODE OF THE INVENTION

The present invention is directed to a folyl extract of fermentedsoybean (folyl EFS) produced by fermenting a culture including a folicacid and soybean extract using a microorganism and an applicationthereof.

γ-polyglutamate is generated when soybeans are fermented using amicroorganism and is a polymer in which an amine group of a glutaminicacid is peptide-linked to γ-carboxyl group. Meanwhile, the folic acid(Vitamin B9 or pteroyl glutamic acid) is a glutamic acid analog in whicha glutamic acid binds to a pteroyl ring. The inventors of the presentinvention identified that y-polyglutamate, which is conventionallyproduced when soybeans are fermented, can be generated by synthesisenzymes produced by a microor-ganism of the genus Bacillus, andconducted experiments based on the fact that when synthesis enzymes arebrought into contact with the folic acid in which the pteroyl ring bindsto the glutamic acid, synthesis enzymes are inclined to polymerize thefolic acid. As a result, the inventors of the present invention foundthat when the folic acid is added when soy-beans are fermented using amicroorganism, the folic acid is incorporated into γ-polyglutamate byenzymes produced by the microorganism and thus, a new type of polymer,that is, folyl polyglutamate including the folic acid and the glutamicacid can be mass-produced. Accordingly, the present inven-tion providesa folyl EFS and a method of mass-producing the folyl EFS. The folyl EFSaccording to the present invention includes folyl polyglutamate. Also,the folyl EFS according to the present invention may include levan(microbial poly-fructan) that is produced when soybeans are fermented.

The folic acid incorporated in the folyl EFS has very differentcharacteristics from a folic acid on its own, and has high solubilitywith respect to water and thus, can be more efficiently used inindustrial applications and more easily absorbed into small bowels ofanimals.

Also, since the folyl EFS according to the present invention may alsoinclude levan (microbial polyfructan) that is produced when soybeans arefermented, growth of bifidus lactobacillus can be promoted and thus,cleaning effect of stomach and intestines and an obesity preventioneffect can be obtained.

The folyl EFS according to the present invention also has a higherimmunostimulating effect and an anti-allergic effect in proportion tothe concentration of the folyl EFS, which has been identified through invitro tests and animal tests. A folic acid itself does not have thoseimmunostimulating and anti-allergy effects. The folyl EFS according tothe present invention inhibits secretion of histamine (see FIGS. 1 and2), and alleviates anaphylaxis (see FIG. 3). Accordingly, the folyl EFSaccording to the present invention can be used in food products forpreventing acute allergy in babies and children and improvingconstitution of adults. Also, the folyl EFS according to the presentinvention can be used in a skin external application or cosmeticcomposition for alleviating atopic dermatitis or allergies.

The folyl EFS according to the present invention promotes biosynthesisof collagen (see FIG. 6), and has a wrinkle-improvement effect (seeTable 5). Accordingly, the folyl EFS according to the present inventioncan be used in moisturizing, skin-improving external applications orcosmetic compositions based on metabolism activation, and has awrinkle-improvement characteristic and effects caused as a resultthereof.

The folyl EFS according to the present invention promotes absorption ofcalcium in small bowels of animals (see FIG. 4). Accordingly, the folylEFS can be used together with a pharmaceutical metal such as calcium,iron, or manganese, and thus various products can be produced including,in addition to functional products, pharmaceutical products, such asnutrition suppliers, anti-anemic agents for pregnant women, or diabetessupplements.

Also, the folyl EFS according to the present invention promotes bonegrowth (see Table 4), and increases growth factors in blood (IGF-10)(see FIG. 5). An example of a currently available growth promotingmaterial is KI-180 which has been co-developed by Highki growth clinicand the Korea Food Research Institute. KI-180 is expensive because it isextracted from herbal medicines and thus is used in expensivepharmaceutical products, not food products. Thus, it is difficult tocommercialize KI-180. However, since the folyl EFS according to thepresent invention can be mass-produced through fermentation with amicroorganism, the folyl EFS can be used in a wide range ofapplications, such as functional foods for children,osteoporosis-prevention-functional foods for postmenopausal women, andnutrition suppliers. Thus, the folyl EFS can be more easilycommercialized than conventional growth promoting materials.

Based on the experimental results described above, the present inventionprovides:

[1] The folyl EFS, including folic acid polyglutamate as a majorcomponent, wherein the folyl EFS is produced by fermenting a cultureincluding a folic acid and soybean extract by using a microorganism;

[2] The folyl EFS of [1], wherein the microorganism is selected from agenus bacillus microorganism or a genus corynebacterium microorganism;

[3] The folyl EFS of [1], wherein the microorganism is Bacillussubtilis;

[4] The folyl EFS of any one of [1] to [3], wherein the folyl EFS has anis anti-histamine effect;

[5] The folyl EFS of any one of [1] to [3], wherein the folyl EFS has ananti-allergic effect;

[6] The folyl EFS of any one of [1] to [3], wherein the folyl EFS has acalcium-absorption-promotion effect;

[7] The folyl EFS of any one of [1] to [3], wherein the folyl EFS has abone-growth-promotion effect;

[8] The folyl EFS of any one of [1] to [3], wherein the folyl EFS has acollagen-biosynthesis-promotion effect;

[9] The folyl EFS of any one of [1] to [3], wherein the folyl EFS has awrinkle-improvement effect;

[10] The folyl EFS of any one of [1] to [3], wherein the folyl EFS has acell-growth-promotion effect;

[11] A composition for skin external application or cosmetics, includingthe folyl EFS of any one of [1] to [3] as an active component;

[12] The composition for skin external application or cosmetics of [11],wherein the composition is used for anti-allergic or improving atopicdermatitis;

[13] The composition for skin external application or cosmetics of [11],wherein the composition is used to activate metabolism of skin cells,prevent formation of wrinkles, or improve the appearance of wrinkles;

[14] The composition for skin external application or cosmetics of [11],wherein the composition is used to inhibit cell damage caused byultra-violet (UV) radiation;

[15] A health supplement food composition, including the folyl EFS ofany one of [1] to [3] as an active component;

[16] The health supplement food composition of [15], wherein the healthsupplement food composition is used in drinks;

[17] The health supplement food composition of [15], wherein the healthsupplement food composition has a growth promotion effect;

[18] The health supplement food composition of [15], wherein the healthsupplement food composition has an osteoporosis prevention effect;

[19] The health supplement food composition of [15], wherein the healthsupplement food composition has a calcium-absorption-promotion effect;

[20] The health supplement food composition of [15], wherein the healthsupplement food composition prevents occurrence of allergy or improvesconstitution;

[21] A feed composition, including the folyl EFS of any one of [1] to[3] as an active component;

[22] A pharmaceutical composition for promoting cell growth, includingthe folyl EFS of any one of [1] to [3] as an active component;

[23] A pharmaceutical composition for treating or preventingosteoporosis, including the folyl EFS of any one of [1] to [3] as anactive component;

[24] A pharmaceutical composition for promoting absorption of calcium,including the folyl EFS of any one of [1] to [3] as an active component;and

[25] A method of manufacturing a folyl extract of fermented soybean(EFS) by fermenting a culture including a folic acid and soybean extractby using a microorganism.

The folyl EFS according to the present invention may be produced usingsoybean extract. A method of producing soybean extract will now bedescribed in detail. Soybeans are added to distilled water, wherein theratio of soybeans to distilled water is about 0.1% to about 50%. Themixture is heated at 100° C. for 1 to 2 hours and slowly cooled andthen, a solid content is removed therefrom, thereby completely producingthe soybean extract. A medium is prepared using 0.1 to 25% of glucose,0.01 to 10% of Na₂HPO₄, and 0.01 to 10% of NaH₂PO₄. Then, 0.1 to 90% ofthe soybean extract and 0.01% to 10% of a folic acid are added to themedium, the resultant medium is sterilized, and then Bacillus subtilisseparated from foods is inoculated into the medium and a fermentationprocess is performed at a temperature of 10 to 60° C., and specificallyof 35 to 55° C., at pH of 5.5 to 8.5, in facultative fermentationconditions, for 48 to 96 hours. After the fermentation process iscomplete, non-soluble materials such as cells are removed throughcontinuous centrifugation and the pH of the resultant solution iscontrolled using 2M HCl to be pH 3 to 5, specifically pH 4. Then,ethanol is added thereto wherein the amount of ethanol is two or moretimes greater than that of the pH-controlled solution. As a result, afolyl EFS is obtained and lyophilized and preserved in powder. Groupingaccording to molecular weight may be performed using a conventionalmethod. For example, the grouping may be performed by chromatography orusing a cross-flow ultrafiltration unit.

A microorganism that can be used to ferment a folic acid and the soybeanextract used in the present invention may be a genus Bacillusmicroorganism or genus Corynebacterium microorganism which is capable ofproducing γ-polyglutamate. Examples of the genus Bacillus microorganisminclude Bacillus subtilis, Bacillus anthracis, Bacillus lichenifonnis,and Bacillus megaterium. For example, the genus Bacillus microorganismmay be Bacillus subtilis. The genus Corynebacterium microorganism may beCorynebacterium glutamicum, but is not limited thereto.

Also, the folyl EFS has an anti-allergic effect and promotes the growthof animal including human and Bifidobacterium and Lactobacillus growth,and thus contributes to high livestock profit and high meat quality.Accordingly, the folyl EFS can be used as feed additives in the animalfeed industry.

A composition according to the present invention may include the folylEFS, wherein the amount of the folyl EFS may be 0.01 to 60 wt. %,specifically 0.1-50 wt. %, of the composition.

In a cosmetic composition according to the present invention, the folylEFS may be used together with appropriate amounts of components that areused in a conventional skin cosmetic material. Examples of suchcomponents include an oil material, water, a surfactant, a moisteningagent, lower alcohol, a thickener, a chelating agent, a colorant, apreservative, and perfumes. The cosmetic composition including the folylEFS according to the present invention may be used in moisturizingcosmetic products, face cleansing products, or other cosmetic products,but is not limited thereto. Specifically, the cosmetic compositionincluding the folyl EFS according to the present invention can be usedin cosmetic products, such as astringent cosmetic water, emollientcosmetic water, nutrient cosmetic water, various creams, essence, packs,or foundations; cleansing products; face cleansing products; soap;treatments; or cosmetic solutions. More specifically, the cosmeticcomposition including the folyl EFS according to the present inventioncan be used in skin lotion, skin softener, skin toner, astringentlotion, milk lotion, moisturizing lotion, nutrient lotion, massagecream, nutrient cream, moisturizing cream, hand cream, essence, nutrientessence, packs, soap, shampoo, cleansing foams, cleansing lotion,cleansing cream, body lotion, body cleanser, oil material, press powder,loose powder, or eye shadow.

A pharmaceutical composition according to the present invention may beused in a solid, semi-solid, solution, or oil form, or in a form of adispersant, a micelle, or a liposome. The pharmaceutical composition mayinclude: an organic or inorganic carrier or an excipient that issuitable for intestinal or non-oral administrations; and the folyl EFSaccording to the present invention as an active component. The activecomponent can be mixed with a conventionally non-toxic pharmaceuticallyacceptable carrier that is suitable for, for example, a tabletformulation, a pellet formulation, a capsule formulation, is asuppository formulation, a solution formulation, an oil formulation, asuspension formulation, an ointment formulation, or any type offormulation that is easily used. An available carrier may be glucose,lactose, gum arabic, gelatin, mannitol, starch paste, magnesium salttrisilicate, talc, corn starch, keratin, colloidal silica, potatostarch, urea, triglyceride having an intermediate chain length, or anytype of carrier that is suitable for manufacturing formulations, whichmay be solid, semi-solid, or liquid. The pharmaceutical composition mayfurther include a supplement, a stabilizer, a thickener, a colorant, ora perfume. The pharmaceutical composition including the active componentmay be formed in any formulation that can be easily orally administered,for example, a tablet formulation, troche, a lozenge-shaped tabletformulation, an aqueous or oil suspension formulation, a dispersiblepowder formulation, a granule formulation, a oil, hard or soft capsuleformulation, a syrup formulation or an elixir formulation. Thesecompositions for oral administration may be prepared using any methodthat is used to manufacture a pharmaceutical composition and known inthe art. To obtain excellent pharmaceutical characteristics and makethese compositions for oral administration more attractive in terms offlavor, the compositions for oral administration may further include atleast one functional agent selected from the group consisting of asweetener such as sucrose, lactose or saccharin; a favoring agent suchas peppermint, pyrola japonica, or cherry; a colorant; and a preservingagent. A tablet including an active component and a non-toxicpharmaceutically acceptable excipient can be produced using aconventional method. The non-toxic pharmaceutically acceptable excipientmay be: a non-active excipient such as calcium carbonate, lactose, acalcium phosphoric acid or a sodium phosphoric acid; a granulating agentor decomposing agent, such as corn starch, potato starch or an alginicacid; a binder such as tragerkans rub, a corn potato, gelatin, orAcasia; or a lubricant such as magnesium stearic acid, stearic acid, ortalc. The tablet may be not coated, or may be coated using aconventional method to delay the decomposition or absorption of thetablet in the gastrointestine and thus to sustain the pharmaceuticaleffect for a longer period of time. A delaying material for this purposemay be glyceryl monostearate or glyceryl distearate. Those materials canalso be used to form an osmotic treatment tablet formulation to controlemission of the active component.

In some cases, an oral formulation may be a hard gelatin capsule inwhich an active component is mixed with a non-active solid diluent, forexample, calcium carbonate, calcium phosphoric acid, or kaolin, or asoft gelatin capsule in which an active component is mixed with water oran oil medium, such as peanut oil, liquid paraffin, or olive oil.

Since subjects to be treated have various symptoms and drugs haveparticular therapeutic characteristics, determining responses ofsubjects with respect to treatments and changing administration dosagesaccording to responses should be conducted by experts. Typically, adaily administration dosage may be about 10 μg to about 200 mg/bodyweight (kg), specifically about 50 μg to about 100 mg/body weight (kg).A daily administration frequency may be four times or less.

A food composition including the folyl EFS according to the presentinvention may be used to produce various foods. Examples of such foodsinclude meats, sausages, bread, chocolates, candies, snacks,confectionery, pizza, various types of noodles, gums, dairy productsincluding ice cream, various soups, beverages, teas, drinks, alcoholicdrinks, and vitamin composites, but the present invention is not limitedthereto.

The present invention will be described in further detail with referenceto the following examples and experimental examples. These examples andexperimental examples are for illustrative purposes only and are notintended to limit the scope of the present invention.

Example 1 Production of Folyl EFS

To produce a folyl extract of fermented soybean (folyl EFS), first,soybean extract was prepared. Specifically, soybeans were added todistilled water, wherein the ratio of soybeans to distilled water was50% and the mixture was heated at 100° C. for 1 hour and slowly cooledand then, a solid content was removed therefrom, thereby producingsoybean extract. A medium was prepared using 2.0% of glucose, 1.0% ofNa₂HPO₄, and 0.5% of NaH₂PO₄. Then, 90% of the soybean extract and 0.5%of the folic acid were added to the medium, the resultant medium wassterilized, and then Bacillus subtilis separated from foods wasinoculated into the medium and a fermentation process was performed at atemperature of 45° C. for 72 hours. After the fermentation process wascomplete, non-soluble materials such as cells were removed throughcontinuous centrifugation and the pH of the resultant solution wascontrolled using 2M HCl to be pH 4. Then, ethanol was added theretowherein the amount of ethanol was two or more times greater than that ofthe pH-controlled solution. As a result, a folyl EFS was obtained andlyophilized and preserved in powder. To produce a polymer having amolecular weight of 1000 KDa, polymers having a molecular weight of 500KDa or less were removed using Sepharose 4B, and the remaining solutionwere divided using a cross-flow ultrafiltration unit (produced byVivascience Co.) into a fraction containing polymers having a molecularweight of 1,000 KDa or more and a fraction containing polymers having amolecular weight of less than 1,000 KDa. The average molecular weight ofthe fraction of polymers having the molecular weight of 1,000 KDa ormore was measured through gel permeation chromatography (GPC) usingliquid chromatography (LC) and the measured average molecular weight was2,500 KDa. These two fractions were dried and used in the same powderamount.

Example 2 Production of Composition Including the Folyl EFS

Various compositions including 0.0 to 10.0 wt. % of the folyl EFSprepared in Example 1 and additives which can be used in foods andmedicines were prepared.

(1) Preparation Example 1 Food Composition Including Folyl EFS

Food compositions that include components and amounts thereof as shownin Table 1 were prepared to produce drinkable health supplement foods.In these compositions, the amount of refined water was appropriatelycontrolled according to the amount of natural preservative.

TABLE 1 Food compositions including folyl EFS (wt. %) Compo- Compo-Compo- Compo- Compo- Compo- Component sition 1 sition 2 sition 3 sition4 sition 5 sition 6 Folyl EFS — — 0.05 0.1 0.2 0.3 prepared in Example 1Cromoline —  1 — — — — sodium salt Cyclodextrin  2  2 2   2   2   2  Trace amounts  3  3 3   3   3   3   of additives such as mineralsDistilled water 95 94 94    94   94   94   Natural appro- appro- appro-appro- appro- appro- preservative priate priate priate priate priatepriate

(2) Preparation Example 2 Cosmetic Composition Including Folyl EFS

Cosmetic compositions that included components and amounts thereof asshown in Table 2 and a control composition including distilled waterinstead of the folyl EFS were formulated. In Table 2, (A) representswater-soluble materials, and (B) represents oil-soluble materials. Theamount of the sterilized distilled water was appropriately controlledaccording to amounts of preservative, flavor material, and pigment.

TABLE 2 Cosmetic compositions including folyl EFS (wt. %) ComponentComposition 1 Composition 2 A Ethanol 5.0 5.0 Folyl EFS prepared inExample 1 — 1.0 Glycerin 4.0 4.0 1,3-butyleneglycol 4.0 4.0 Sterilizeddistilled water 68.5 67.5 B Ethanol 5.0 5.0 Cetyl alcohol 1.0 1.0 Beeswax 0.5 0.5 Vaseline 2.0 2.0 Squalene 6.0 6.0 Dimethylpolysiloxane 2.02.0 POE(10)monooleic acid Ester 1.0 1.0 Glycerol monostearinic acidEster 1.0 1.0 Preservative appropriate appropriate Flavor materialappropriate appropriate Pigment appropriate appropriate

(3) Preparation Example 3 Feed Composition Including Folyl EFS

Feed compositions that included components and amounts thereof as shownin Table 3 were prepared.

TABLE 3 Feed compositions including folyl EFS (wt. %) CompositionComposition Component 1 2 Folyl EFS prepared in Example 1 —  1 Calciumcarbonate 70 70 zeolite 30 29

Example 3 Anti-Allergy Test Example 3-1 Anti-Histaminic EffectIdentified by Observing Mast Cells Through Microscopy

To identify the anti-histamine effect of the folyl EFS, a test materialwas applied to mast cells (ATCC TIB-64, P815) and observed the mastcells through microscopy (see George Chiang et al, The Journal ofUrology, December, 164, 2000, p 2119˜2125). FIG. 1A shows an image ofnormal mast cells, FIG. 1B shows an image of mast cells which weretreated with 10 ug/ml of C48/80(sigma) as a stimulant and placed leftfor 30 minutes, and FIG. 1C shows an image of mast cells which weretreated with the folyl EFS and the stimulant, and placed left for 30minutes. Referring to FIGS. 1A to 1C, normal mast cells were round andsmooth. Mast cells were rough when an immune reaction occurred. However,when mast cells were treated with C48/80 as a stimulant and placed leftfor 15 minutes, mast cells were distorted and had protrusions onsurfaces thereof, and when 30 minutes had passed, mast cells completelylost their original shapes and burst, thereby secreting histamine. Thedistortion of surfaces of mast cells is a phenomenon in which varioussignal delivery materials are secreted from cell membranes of mast cellsstimulated by C48/80 and stability of cell membranes is degraded, andthus surfaces of the cell membranes are unstable. Accordingly, theanti-histamine effect can be identified based on how well the originalsurface shapes of mast cells are maintained after the C48/80 treatment.However, when treated with the folyl EFS and placed left for 15 minutes,mast cells retained their original shapes, and even when 30 minutes hadpassed, only a small number of the cells secreted histamine and cellmembranes thereof burst.

Example 3-2 Anti-Histaminic Effect Identified by Quantifying Histamineof Mast Cells

This experiment was performed to identify how much the folyl EFS reducesthe amount of histamine secreted when mast cells are treated with astimulant. The amount of histamine secreted was quantified using ELISA.1% sodium cromolyn, which is known as an asthma treatment drug, was usedas a control group, and C48/80 was used as a stimulator to induceactivation of mast cells (ATCC TIB-64, P815). FIG. 2 is a bar chartshowing the results of the experiment.

Referring to FIG. 2, when about 0.5% of the sodium cromolyn as an asthmato treatment drug was used, a histamine secretion inhibition rate was50% and thus, an ED50 value was 0.5%, and when the folyl EFS was used,an ED50 value was about 0.063%, 8 times lower than that when the sodiumcromolyn was used. As described above, it can be seen that the folyl EFShas an excellent anti-histamine (anti-allergic) effect.

Example 3-3 Anti-Allergic (Anti-Anaphylaxis) Tests

To confirm the anti-histamine effect of the folyl EFS, which has beenidentified in Example 3-1 in animals, mice were fed Compositions 1-6used in Preparation Example 1 for 7 days and then, C48/80 wasintraperperitoneally injected thereto to induce anti-anaphylaxis. FIG. 3is a bar chart showing the results of the experiment.

C48/80 is a material that stimulates immune organs and induces acuteanaphylaxis within 30 minutes when intraperperitoneally injected. In thecurrent experiment, the number of test animals which died within apredetermined time period (24 hours) after the intraperperitonealinjection of the C48/80 was counted. In the current experiment, 10 mice(BALB/C, male) were used per group, and each group was tested for threetimes and the average value of the three experimental results wasrepresented as a death ratio (%).

Referring to FIG. 3, the death rate of a group fed with Composition 1 towhich nothing was added was 93.3% and 9 out of 10 mice died within 24hours after C48/80 was injected. The death rate of a group fed withComposition 2 to which the sodium cromolyn was added, that is, the deathrate of the control group, was 20%. Experimental groups wererespectively fed with Compositions 3 to 6 to which the folyl EFS havingvarious concentrations of 0.05 to 0.3% were added. The death rate of theexperimental group of Composition 3 to which 0.05% of the folyl EFS wasadded was 33.3%, the death rate of Composition 4 was 20%, the death rateof Composition 5 was 6.7%, and the death rate of Composition 6 was16.7%. In consideration of these results, it can be seen that 1% sodiumcromolyn as an asthma treatment drug inhibits development of anaphylaxisup to 80% and the folyl EFS inhibits development of anaphylaxis up to94% at the concentration of 0.2%. However, the mice which survivedwithin 24 hours after the stimulus C48/80 was injected developedabnormally swollen anuses. Such incident was found in about 80% of themice to which Composition 2 including sodium cromolyn, and about 10%,0%, 0%, and 0% when Compositions 3 through 6 including variousconcentrations of folyl EFS were respectively used. Accordingly, it canbe seen that sodium cromolyn inhibited development of anaphylaxis andprevented death of mice but did not inhibit sequela of anaphylaxis, andthe folyl EFS inhibited the sequela of anaphylaxis as well as death ofmice.

Example 4 Calcium Absorption Promotion Effect

The current experiment was performed using mice (BALB/C, male) and eachfeed was orally administered to mice two times per day. The oraladministration period was 3 days and after the oral administrationperiod, small bowels of the mice were isolated. The obtained smallbowels were washed with saline 3 or more times, and tissues of the smallbowels isolated from each group were ground with a teflon homogenizer.The obtained product was centrifuged and the supernatant was isolated.Then, the amount of calcium absorbed into the small bowels was measuredusing a calcium quantification kit (Wako Co.). FIG. 4 is a bar chartshowing the results of the experiment.

Referring to FIG. 4, a group given with the folyl EFS added feed hadabout 84 to 96% increase of a calcium absorption rate compared to acontrol group given nothing added feed. A calcium absorption rate of agroup given calcium salt added feed was about 4.7%, a group given caseinphosphopeptide (CPP) added feed was about 21%, and a group givenγ-polyglutamate added feed was about 56.8%. For the folyl EFS, when themolecular weight was 500 to 1,000 KDa, the calcium absorption rate washigher than when the molecular weight was higher than 1,000 KDa. Such aresult may be because when the molecular weight of the folyl EFS is toohigh, a calcium ion, that is a bi-valent ion, reacts with the folyl EFShaving a negative charge and thus, non-soluble precipitate may beproduced. Accordingly, it is desirable that a calcium-folyl EFScomposite is produced to be soluble when the molecular weight of thefolyl EFS is high.

Example 5 Growth Promotion Effect

Since, as described in Example 3, the folyl EFS promoted absorption ofcalcium into bowels, the current experiment was performed to identifywhether the calcium absorbed into bowels affects animal bone growth andgrowth factors in blood (IGF-1). Mammals were given folyl EFS added feedfor two weeks, and the length and weight of femurs (thigh bones) of themammals were measured and blood was taken to measure IGF-1 that is thegrowth factor. The results were compared with those of a control groupgiven saline added feed. The tested mammals were four-week old rats (SDrat, male). A comparative group was given “Kikerup gold” productcontaining as a major to component herb extracts of Acantopanax, a redflower seed, Rehmanniae radix preparata, Poria cocos, Dioscoreaerhizoma, Corni fructus, antler etc., which is produced by a P&B company,and tested under the same condition as when the folyl EFS was used.

The results are shown in Table 4 and FIG. 5.

When the standard error was 5% or less, the femurs (thigh bones) of thecontrol group fed with saline had an average length of 29.22 mm, thefemurs (thigh bones) of the comparative group fed with the growthpromotion product of the P&B company had an average length of 29.63 mm,and the femurs (thigh bones) of the experimental group fed with thefolyl EFS had an average length of 30.04 mm. Compared to the controlgroup, the comparative group had a growth promotion effect of about 1.4%and the experimental group had a growth promotion effect of about 3.1%(Table 4). IGF-1, which is known as a growth promotion factor in blood,was quantified by ELISA. As a result, the control group had 1409.5 μg/mlof IGF-1, the comparative group had 1566 μg/ml of IGF-1, and theexperimental group had 1637.1 μg/ml of IGF-1. That is, IGF-1 of thecomparative group was 11.1% higher than IGF-1 of the control group, andIGF-1 of the experimental group was 16.2% higher than IGF-1 of thecontrol group (FIG. 5). The weights of the femurs (thigh bones) of thecomparative group and experimental group were about 1.3% and about 2.3%higher than that of the control group, respectively. In consideration ofthose results, it can be seen that the folyl EFS has a growth promotioneffect in animals. In addition, when the growth promotion product of theP&B company is compared with the folyl EFS in terms of manufacturingcost, the manufacturing costs of the folyl EFS are about 1/20 of thegrowth promotion product (Kikerup gold) of the P&B considering that thegrowth promotion product contains vitamins and nutrients. Accordingly,it is assumed that the folyl EFS has excellent characteristics in termsof various aspects including the manufacturing cost.

TABLE 4 Bone growth promotion effect of folic acid Upper Middle BottomTest Length (mm) Width (mm) Width (mm) Width (mm) Weight (mg) Control29.22 ± 0.31 6.20 ± 0.07 2.43 ± 0.03* 3.64 ± 0.03* 307.2 ± 9.1 Kikerupgold (P&B) 29.63 ± 0.15 6.24 ± 0.05 2.31 ± 0.03   3.50 ± 0.05   311.2 ±9.6 Folyl EFS   30.14 ± 0.16* 6.22 ± 0.12 2.31 ± 0.03   3.55 ± 0.05  314.3 ± 7.6 *P < 0.05, Ducan's multiple test

Example 6 Wrinkle-Improvement Effect and Wound-Healing Effect Example6-1 Test Regarding Cell Activation and Collagen Biosynthesis Promotion

The inventors of the present invention found that the folyl EFS has ahigh cell activation effect. The meaning of the term ‘cell activationeffect’ includes activation of to cell growth, activation of productionof metabolites, activation of mitochondria in cells for detoxification,etc. The cell activation effect may induce a skin-wound healing effectand a wrinkle-improvement effect.

It was identified through the current experiment that the folyl EFSpromotes cell growth and collagen biosynthesis. A cell line used in thecurrent experiment was a primary culture of a fibroblast (human dermalfibroblast neonatal; (HDF-N; Gibco, Cooper, M. L. et al, Biomaterials12:243, 1991), and a culture media used was a fibroblast neonatal growthmedium (FGM, KDR Co.). Since the folyl EFS included polyglutamate as amajor component in which about 10% of folic acid was incorporated, thefolyl EFS was compared with a normal folic acid. 1×10⁵ cells/well of thehuman dermal fibroblast neonatal and a folic acid or the folyl EFS wereincubated for 24 hours, the incubation products were photographed (seeFIG. 6), and viable cells were quantified through neutral red (NR) assay(Sigma; Shao-Zeng Z. et al., Cell Biology and Toxicology, 6:2, 1990)(see FIG. 7).

When the folyl EFS was used, the cell growth rate was high, andspecifically, when the same concentration of folic acid was used, ahigher cell growth rate could be obtained than when only a folic acidwas used. In addition, when only a folic acid was used and theconcentration of the folic acid was gradually increased, the cell growthrate was not increased accordingly. However, when the folyl EFS was usedand the concentration of the folyl EFS was gradually increased, the cellgrowth rate was distinctively changed accordingly. Such results may stemfrom a difference in water solubility of the folic acid and the folylEFS. Specifically, the folyl EFS has high water solubility and thus morecells are affected. Such cell growth promotion leads to awrinkle-improvement effect when applied in cosmetic products and awound-healing promotion effect when applied in pharmaceutical products

The folyl EFS and the human dermal fibroblast neonatal were incubatedfor 3 days, and the supernatant was isolated and collagen was quantifiedusing a human collagen type-1 ELISA kit (Cosmo Bio Co.). A sample usedin the current experiment was a 1% solution containing Composition 2used in Preparation Example 2, and 4% of 1,3-butyleneglycol was added tothe sample and preserved. Also, to compare with the effect of the folylEFS, a folic acid solution in which the concentration of the folic acidwas the same as that in the folyl EFS was prepared. In addition, 10ng/ml of transforming growth factor-beta (TGF-β; Sigma), which is knownas a promotion factor for collagen biosynthesis, was compared with thefolyl EFS.

The collagen biosynthesis promotion effect of the folyl EFS is shown inthe bar chart of FIG. 8. A folyl EFS produced as a cosmetic sourcematerial was a 1% liquid product. In FIG. 8, the concentration (%) ofthe x-axis denotes a ratio of the volume of the folyl EFS to the volumeof the entire culture solution (v/v, %). The results are represented asthe ratio of the value of collagen of each group with respect to theaverage value of collagen in a group not treated with anything when theaverage value of collagen in the group not treated with anything was setat 100%. As a result, for TGF-β, the maximum promotion rate was 22% at0.63% (v/v, %), and for the folyl EFS, the maximum promotion rate was109% at 0.63%(v/v, %). That is, the promotion rate of the folyl EFS wasfour times higher than that of TGF-β applied to the comparative group,which shows that a wrinkle-improvement effect can be obtained throughexcess production of collagen in human skin. Meanwhile, when only afolic acid was used, a collagen biosynthesis promotion effect did notoccur within an error range. Accordingly, it can be seen that the folylEFS can be very effectively used as a cosmetic material, specifically, amaterial for improving the appearance of wrinkles by collagenbiosynthesis.

Example 6-2 Test Regarding Wrinkle-Improvement Effect of CosmeticComposition

To identify a wrinkle-improvement effect of the folyl EFS, Composition 1(control group) prepared in Example 2 and Composition 2 (experimentalgroup) prepared in Preparation Example 2 were applied to wrinklessurrounding the eyes of 29 women who were 30 year olds two times per dayfor one month. After one month, the degree of wrinkle appearanceimprovement was evaluated by referring to questionnaires answered by thesubjects and analysis results regarding nutrients of wrinkles. For thequestionnaires answered by the subjects, four selective answers wereprovided to each question, ‘no improvement’, ‘small improvement’,‘significant improvement’, and ‘very significant improvement,’ whencompared before and after the application.

Wrinkles were evaluated through image analysis. Before the test wasperformed, replicas below the eyes were collected using Visioscan SV600(CK Co.), and when the test was finished, replicas were collected fromthe same position to measure wrinkle density through image analysis. Theresults are represented as a wrinkle decrease rate

The results are shown in Table 5, and it can be seen that whenComposition 2 including the folyl EFS was compared with Composition 1,wrinkles were alleviated and the appearance thereof was improved. Inaddition, when Composition 2 including the folyl EFS was applied, thewrinkle density was reduced to about 45%, compared to that before theapplication.

Such results show that when the composition according to the presentinvention is used as a skin external application, the appearance ofwrinkles formed in the skin can be effectively improved. Accordingly, itcan be seen that the folyl EFS can be effectively used as a cosmeticmaterial.

TABLE 5 Improvement degree and density of skin wrinkles caused bycosmetic compositions No Small Significant Very improve- improve-improve- significant Wrinkle ment ment ment improvement density (people)(people) (people) (people) (%) Composition 25 3  1  0 95 1 Composition 0 4 10 15 45 2

Example 7 Effect of Inhibiting Cell Damage Caused by Ultra-Violet (UV)Radiation

The inventors of the present invention identified in the currentexperiment that the folyl EFS inhibits cell damage caused by UVradiation.

1×10⁵ cells/well of a primary culture of human keratinocyte wasincubated in a human keratinocyte growth medium (KGM, KDR Co.) togetherwith a folyl EFS in which the amount of a folic acid was about 10 wt %for about 72 hours, and then UV rays having a wavelength of 306 nm wereirradiated to the grown-cells for a predetermined time period. Theconcentration of folic acid in a final product can be controlled byadjusting the concentration of the folic acid incorporated in the folylEFS during fermentation. After UV rays were irradiated, the resultantculture was incubated for about 24 hours and viable cells werequantified through an NR assay. The irradiation time period was definedas a time period in which about 50% of the cells died in a preliminaryexperiment. The results are interpreted as follows:Survival rate(%)=(growth rate of each group after UV rays wereirradiated)/(growth rate of the group having the same concentrationwhich was not exposed to UV radiation).

In media in which folyl EFSs having various concentrations were grown,UV radiation-induced cell damage was inhibited in proportion to theconcentration of grown cells. Unlike the results of the cell growthpromotion test in Example 6, the highest survival rate was obtained whenthe concentration of the folic acid was 0.62%. Such a result may bebecause although the growth rate of cells grown in a medium includingabout 0.62% of the folyl EFS was lower than that of the growth rate ofcells grown in a medium including about 0.31% of the folyl EFS, whenabout 0.62% of the folyl EFS is used, the UV induced cell damageinhibition rate was higher than when about 0.31% of the folyl EFS wasused. Accordingly, it can be seen that when the folyl EFS is added tocosmetic products, ultra-violet (UV) radiation is effectively blocked.Specifically, use of about 0.1-6% of the folyl EFS contributes to anincrease in the UV blocking effect in regular formulations, such aslotion and cream, in addition to sun-blocking products.

The invention claimed is:
 1. A method of using a composition comprisinga folyl extract of fermented soybeans (EFS) for external skinapplication, comprising the step of administering the composition ontoskin of a subject, wherein the composition promotes cell growth,promotes biosynthesis of collagen, or improves the appearance ofwrinkles, and wherein the folyl EFS comprises folyl polyglutamatepolymer, where folic acid is incorporated into the polyglutamate, as amajor component, and the folyl EFS is produced by fermenting a culturecomprising 0.1 to 90% of soybean extract and 0.01 to 10% of additionallyadded folic acid using a microorganism capable of producing the folylpolyglutamate polymer and removing non-soluble materials to obtain theEFS.
 2. The method of claim 1 wherein the composition comprisesapproximately 0.1 to 6% folyl EFS by weight.
 3. The method of claim 1wherein the composition further comprises water, an oil, an alcohol anda surfactant.
 4. A method of using a composition comprising a folylextract of fermented soybeans for external skin application comprisingthe step of administering the composition onto skin of a subject,wherein the composition inhibits cell damage caused by ultra-violet (UV)radiation, and wherein the folyl EFS comprises folyl polyglutamatepolymer, where folic acid is incorporated into the polyglutamate, as amajor component, and the folyl EFS is produced by fermenting a culturecomprising 0.1 to 90% of and soybean extract and 0.01 to 10% ofadditionally added folic acid using a microorganism capable of producingthe folyl polyglutamate polymer and removing non-soluble materials toobtain the EFS.
 5. The method of claim 4 wherein the compositioncomprises approximately 0.1 to 6% folyl EFS by weight.
 6. The method ofclaim 4 wherein the composition further comprises water, an oil, analcohol and a surfactant.